Parathyroid hormone (PTH) is a secreted, protein product of mammalian parathyroid glands that regulates calcium homeostasis through its action on various tissues, including bone and vascular tissue. Research into the physiological role of PTH has identified clinically relevant effects on bone metabolism, there being some clinical evidence that PTH may be useful in the treatment of osteoporosis and related osteopenic afflictions. An effect on vascular tissue and on keratinocyte growth has also been noted.
To obtain PTH in the amounts required for clinical investigations and for commercial purposes, recombinant DNA-based techniques have been successfully applied in the production of such mammalian PTH species as human PTH, bovine PTH, porcine PTH and rat PTH, which in their mature form all contain 84 amino acids arranged in a species-specific sequence. An understanding of the structure of these proteins, and particularly human PTH and bovine PTH, has lead also to the discovery that PTH activity can be attributed to the first 34 N-terminal residues of the mature hormone. This has allowed for the production of biologically active PTH fragments using the solid phase technique of peptide synthesis, to meet PTH demand.
In the interest of furthering development of PTH as a pharmaceutical product, it would be desirable to provide stability-enhanced forms of PTH that are better suited for human administration. It is known from studies with serum-derived PTH samples, and from experience with recombinant PTH production, that the hormone is particularly vulnerable to protease digestion. PTH production in yeast, for example, has shown that PTH is degraded by the yeast-produced kex enzyme, which recognizes dibasic residues. When the lysine residue at position 26 of PTH is replaced with glutamine, however, proteolytic degradation of PTH by the yeast is reportedly reduced (see Reppe et al, J. Biol. Chem., 1991, 266:14198). Other studies targetting this general region of the PTH molecule have indicated that amino acid replacement can cause significant decline in PTH activity. Conversion of the lysine at position 27 for example, resulted in a marked decline in PTH activity (see Wingender et al, WO 90/10067) as did derivatization of the arginine residues at positions 25 and 20 (sse Rosenblatt et al, 1978, Biochemistry, 17:3188).
It is an object of the present invention to provide a novel variant of parathyroid hormone.
It is another object of the present invention to provide a parathyroid hormone variant that exhibits improved stability in the presence of proteolytic enzymes, especially trypsin and trypsin-like enzymes.
It is another object of the present invention to provide a parathyroid hormone variant that exhibits both improved stability in the presence of proteolytic enzymes and a bioactivity that is comparable to parathyroid hormone.
It is another object of the present invention to provide a pharmaceutical composition comprising a parathyroid hormone variant exhibiting improved stability and comparable activity, for use in therapeutic applications.
It is another object of the present invention to provide a process for producing a stability-enhanced variant of parathyroid hormone.